Western blot fail, time and time again

Hey,
I`m really at the end of my Latin. I had enough of my constant fails with WB.
I`m working with sheep lung tissue which I homogenize in a buffer containing protease-inhibitors.
I do a BCA-test, get nice protein concentrations and start WB.
My sample concentrations range from 20ug to 70ug which I dilute in water and loading buffer (DTT, reduced) which
I then incubate for 5min at 95C.
I next do a standard gelelektroforesis (gel 15%agarose) for 1,5h 100V
Next I blot my gel on a nitrocellulose membrane for 45min, 100V.
I block and incubate with my primary antibody overnight.
Next day wash, sec antibody (Odessey), wash and scan with Odessey.
I`ve tested this with several antibodies. First it did not work but I tought it was the loading buffer, changed it.
Then i did a beta-actin WB just as control and it worked. Next I proceeded again with my other antibodies (which I know work on my sheep lung material)
and again nothing. The ladder comes out nicely, but no band for the samples, nothing, not even a smear.
What should I do? I do not see where my mistake could be. Can you please help me?

well, I first thought I had the solution.
I made a calculation error and diluted my samples 100x to much.
So I repeated the experiment for the detection of p-smad in my lung tissue (which worked fine on IHC),
now taking along a positive control (cell lysate stimulated with tgf-beta).
I checked that the samples I took along were samples that showed lots of signal in the IHC.
Again, I did not get a signal for my samples but it did for my positive control.
So either, there is something wrong with my samples, or the Ab does not work on my samples for Western blot ( is that possible, considering that it worked for IHC??)
What I saw is that there is still a pellet left in my sample lysates so the supernatant was not transferred to a fresh tube.
Can that have an effect?
I wonder now to change my lysis buffer. Is that perhaps an option?

well, I first thought I had the solution. I made a calculation error and diluted my samples 100x to much. So I repeated the experiment for the detection of p-smad in my lung tissue (which worked fine on IHC), now taking along a positive control (cell lysate stimulated with tgf-beta).I checked that the samples I took along were samples that showed lots of signal in the IHC. Again, I did not get a signal for my samples but it did for my positive control. So either, there is something wrong with my samples, or the Ab does not work on my samples for Western blot ( is that possible, considering that it worked for IHC??) What I saw is that there is still a pellet left in my sample lysates so the supernatant was not transferred to a fresh tube.Can that have an effect? I wonder now to change my lysis buffer. Is that perhaps an option?

The fact that you are getting signal for the positive control but not your samples would indicate that the samples are the problem and not the antibody. Do you ponceau stain the membrane after transfer? You should do this to see if your samples are separating well and that you are loading enough protein. Why are you diluting your samples with water? This throws off the buffer concentrations. I would dilute in the same buffer you are homogenizing with.

I agree with mdfenko -- it sounds like denaturing your protein results in the loss of the epitope(s) your antibody recognizes. Is this a monoclonal Ab, or are you using polyclonal sera? Try a dot-blot of your samples and see if your Ab recognizes them if they're not denatured (just spot a small amount of each along with a positive and negative control on a suitable membrane, and process the membrane as you would a western).

In the dot-blot approach: What should I do exactly?
Just drop some drops of my protein solution onto the membrane which i use for western blot, then block, incubate Ab and
visualize? No buffer in my proteins?

In the dot-blot approach: What should I do exactly?Just drop some drops of my protein solution onto the membrane which i use for western blot, then block, incubate Ab and visualize? No buffer in my proteins?

yes

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